Oxidative activation of protein kinase Cgamma through the C1 domain. Effects on gap junctions

The accumulation of reactive oxygen species (ROS, for example H2O2) is linked to several chronic pathologies, including cancer and cardiovascular and neurodegenerative diseases (Gate, L., Paul, J., Ba, G. N., Tew, K. D., and Tapiero, H. (1999) Biomed. Pharmacother. 53, 169-180). Protein kinase C (PKC) gamma is a unique isoform of PKC that is found in neuronal cells and eye tissues. This isoform is activated by ROS such as H2O2. Mutations (H101Y, G118D, S119P, and G128D) in the PKCgamma Cys-rich C1B domain caused a form of dominant non-episodic cerebellar ataxia in humans (Chen, D.-H., Brkanac, Z., Verlinde, C. L. M. J., Tan, X.-J., Bylenok, L., Nochli, D., Matsushita, M., Lipe, H., Wolff, J., Fernandez, M., Cimino, P. J., Bird, T. D., and Raskind, W. H. (2003) Am. J. Hum. Genet. 72, 839-849; van de Warrenburg, B. P. C., Verbeek, D. S., Piersma, S. J., Hennekam, F. A. M., Pearson, P. L., Knoers, N. V. A. M., Kremer, H. P. H., and Sinke, R. J. (2003) Neurology 61, 1760-1765). This could be due to a failure of the mutant PKCgamma proteins to be activated by ROS and to subsequently inhibit gap junctions. The purpose of this study was to demonstrate the cellular mechanism of activation of PKCgamma by H2O2 and the resultant effects on gap junction activity. H2O2 stimulated PKCgamma enzyme activity independently of elevations in cellular diacylglycerol, the natural PKC activator. Okadaic acid, a phosphatase inhibitor, did not affect H2O2-stimulated PKCgamma activity, indicating that dephosphorylation was not involved. The reductant, dithiothreitol, abolished the effects of H2O2, suggesting a direct oxidation of PKCgamma at the Cys-rich C1 domain. H2O2 induced the C1 domain of PKCgamma to translocate to plasma membranes, whereas the C2 domain did not. Direct effects of H2O2 on PKCgamma were demonstrated using two-dimensional SDS-PAGE. Results demonstrated that PKCgamma formed disulfide bonds in response to H2O2. H2O2-activated PKCgamma was targeted into caveolin-1- and connexin 43-containing lipid rafts, and the PKCgamma phosphorylated the connexin 43 gap junction proteins on Ser-368. This resulted in disassembly of connexin 43 gap junction plaques and decreased gap junction activity. Results suggested that H2O2 caused oxidation of the C1 domain, activation of the PKCgamma, and inhibition of gap junctions. This inhibition of gap junctions could provide a protection to cells against oxidative stress.     

Endothelial mediators and communication through vascular gap junctions

Cellular interaction in vessels is achieved by multiple communication pathways, including gap junctions (GJs). They provide intercellular channels, allowing direct interaction of endothelial and smooth muscle cells and the coordination of cellular behaviour along the vessel. The latter is a prerequisite for large flow increases because an adaptation of resistance along the vessel length is required. Longitudinal communication is studied by confined local stimulation of arterioles and the observation of responses at distant locations. Certain vascular stimuli induce local and concomitant remote responses of a similar type, verifying rapid longitudinal conduction of vasomotor signals, most likely changes in membrane potential. This is achieved for dilatory responses via the endothelium, possibly by an endothelium-derived hyperpolarising factor (EDHF) that induces local hyperpolarisation, which is then transferred to remote sites through GJs. In vessels, GJs are composed of different connexins (Cx), but Cx40 is of special importance because its lack impairs longitudinal conduction of vasodilations. Interestingly, Cx40-deficient mice are hypertensive, suggesting that Cx40-dependent coupling is necessary to regulate vascular behaviour and peripheral resistance. While the role of other connexins is less well established, an abundance of data has proven the necessity of GJ communication to coordinate vascular behaviour during blood flow regulation.     

Differential regulation of intracellular calcium oscillations by mitochondria and gap junctions

Fluctuations of intracellular Ca²⁺ ([Ca²⁺]_i) regulate a variety of cellular functions. The classical Ca²⁺ transport pathways in the endoplasmic reticulum (ER) and plasma membrane are essential to [Ca²⁺]_i oscillations. Although mitochondria have recently been shown to absorb and release Ca²⁺ during G protein-coupled receptor (GPCR) activation, the role of mitochondria in [Ca²⁺]_i oscillations remains to be elucidated. Using fluo-3-loaded human teratocarcinoma NT2 cells, we investigated the regulation of [Ca²⁺]_i oscillations by mitochondria. Both the muscarinic GPCR agonist carbachol and the ER Ca²⁺-adenosine triphosphate inhibitor thapsigargin (Tg) induced [Ca²⁺]_i oscillations in NT2 cells. The [Ca²⁺]_i oscillations induced by carbachol were unsynchronized among individual NT2 cells; in contrast, Tg-induced oscillations were synchronized. Inhibition of mitochondrial functions with either mitochondrial blockers or depletion of mitochondrial DNA eliminated carbachol—but not Tg-induced [Ca²⁺]_i oscillations. Furthermore, carbachol-induced [Ca²⁺]_i oscillations were partially restored to mitochondrial DNA-depleted NT2 cells by introduction of exogenous mitochondria. Treatment of NT2 cells with gap junction blockers prevented Tg-induced but not carbachol-induced [Ca²⁺]_i oscillations. These data suggest that the distinct patterns of [Ca²⁺]_i oscillations induced by GPCR and Tg are differentially modulated by mitochondria and gap junctions.     

Single-cell measurements of ion concentrations within the intracellular parasite.

Rapid progress has been made in the study of intracellular ion activities of eukaryotic cells through the recent combination of high-resolution microscopy with fluorimetric ion-specific probes. This technique allows a specific ion concentration within a single living cell to be monitored on-line with high temporal and spatial resolution. In this report, Stefan Wünsch, Paul Horrocks, Michael Gekle and Michael Lanzer evaluate the application of single-cell fluorimetry to the study of transport processes in Plasmodium falciparum.     

Measurement of intracellular potassium ion concentrations by n.m.r

39K n.m.r. was used to detect and quantify K+ within human erythrocytes. A shift reagent consisting of an anionic complex of dysprosium(III) with sodium tripolyphosphate permitted a distinction to be made between K+ inside and outside erythrocytes. Intracellular K+ concentrations determined by this method were similar to values obtained by flame photometry.     

A mechanism for isotonic fluid flow through the tight junctions of Necturus gallbladder epithelium

During isotonic fluid flow, Necturus gallbladder epithelium mediates net fluxes of paracellular probes by a convective process. We show here that the paracellular system is modeled by permeation through three populations of channels: (i) convective parallel-sided ones of width 7.7 nm (ii) small diffusive ones of radius 0.6 nm, and (ii) large diffusive ones of radius exceeding 50 nm. The reflexion coefficient of the convective channels is very low and the calculated osmotic flow rate is close to zero when compared with the observed fluid absorptive rate of 2 x 10^–6 cm/sec. Analysis reveals that the convective channels behave as though closed to back-diffusion of probes; if this is due to solvent drag then very high fluid velocities are required, acting through minute areas. There are no transjunctional gradients that could drive the flow, and so the fluid must be propelled through the channel by components of the junction.We propose a mechanism based upon an active junctional peristalsis which allows discrimination on the basis of molecular size, in which the channels are always occluded at some point and so back-diffusion cannot occur. There is no local gradient of salt distal to the junctions and therefore the osmotic permeability of the membranes is irrelevant. High fluid velocities are not required, and the flow can occur over a substantial fraction of the junction. The mechanism must involve motile and contractile elements associated with the junction for which there is already considerable evidence.Symbols A _i filtration area of channel i;i=b (big), s (small) and c (convectional) - B constant for streamline flow - C _i concentration of probe at i - D diffusion coefficient - D ^o diffusion coefficient in free solution - d width of junction - F _i diffusive drag factor in channel i - g ionic conductivity - G _i convective drag factor in channel i - J _ij probe flux from i to j - J _net net probe flux - J _v volume flow per cm² of epithelium - l linear extent of junction per cm² epithelial plane - L length of junctional channel - L _p hydraulic conductivity - N Avogadro's number - q available filtration area fraction of channel - r _s probe molecular radius - r _c channel radius or half-width - S _i steric factor in channel i - V _w,s partial molar volume of water or salt - v _i fluid velocity in channel i - _w dynamic viscosity of water - specific conductivity - ratio of solute radius to channel radius or half-width - diffusive/pressure-driven flow ratio - reflexion coefficient     

Permeability properties and intracellular ion concentrations of epithelial cells in rat duodenum.

Effects of the K+ concentration in the bathing fluid ([K+]l) on the intracellular K+, Na+ and Cl- concentrations ([K+]i [Na+]i and [Cl-]i) as well as on the electrical potential were studied in rat duodenum. Changes in the mucosal K+ concentration ([K+]m), bringing the sum of Na+ and K+ concentrations to 147.2 mM constant, had little effect on the transmural potential difference (PDt), but did induce marked changes in the mucosal membrane potential (Vm). As [K+]m increased, Vm was depolarized gradually and obeyed the Nernst equation for a potassium electrode in the range of [K+]m greater than approx. 60 mM. Experiments of ion analyses were carried out on strips of duodenum to determine the effect of changing the external K+ concentrations on [K+] i, [Na+]i and [Cl-]i. An increase in [K+]o resulted in increases in [K+]i and [Cl-]i and a decrease in [Na+]i, [K+]i approaching its maximum at [K+]o greater than 70 mM. Such changes in [K+]i and [Na+]i seem to correlate quantitatively with the changes in [K+]o and [Na+]o. The values of the ratio of permeability coefficients, Pna+/PK+ were estimated using the Vm values and intracellular ion concentrations measured in these experiments. The results suggested that there appeared a rather abrupt increase in the PNa+/PK+ ratio from 0 to approx. 0.1, as [K+]m decreased.     

NBT-II carcinoma behaviour is not dependent on cell-cell communication through gap junctions

To study the mechanism(s) underlying the proliferation of heterogeneous cell populations within a solid tumour, the NBT-II rat bladder carcinoma system was used. It has been first investigated whether the different cell populations are coupled through gap junctions (GJIC). Cells overexpressing the Cx43 were generated to test for any tumour suppressive activity in vivo. To determine whether GJIC is essential for tumour proliferation and the establishment of a cooperative community effect, NBT-II cells that are incompetent for cell coupling were generated. The data report that (i) carcinoma cells expressing or not FGF-1 are coupled through GJIC in vitro and in coculture and express the gap junction protein Cx43, (ii) overexpression of Cx43 in these cells does not affect their in vitro coupling capacities and in vivo tumourigenic growth properties, (iii) inhibition of GJIC through antisense strategy has no in vivo obvious consequence on the tumour growth properties of the carcinoma, and (iv) the community effect between two carcinoma cell populations does not critically involve cell coupling through gap junctions.     

Steady-state ion transport by nonactin and trinactin.

The steady-state fluxes of Na+, K+, and NH4+ carried by nonactin and trinactin across thin lipid membranes have been measured as functions of ion activity, carrier concentration, and the applied potential. In agreement with earlier studies the conductance, G(O), is found to be proportional to the carrier concentration and, for low activities, to the ion activity. The determination of the dependence of G(O) on activity at high activities is, however, apparently obscured by changes in the concentration of carrier in the membrane. Using the values for the rate constants at zero potential which were determined in the preceding paper, it is possible to adjust the potential dependence of the constants so as to achieve a reasonable fit to the current-voltage relations. The data presented provide further evidence that a single molecule of nonactin or trinactin acts cyclicly as a carrier of univalent cations.     

Steady-state pleural fluid flow and pressure and the effects of lung buoyancy

Both theoretical and experimental studies of pleural fluid dynamics and lung buoyancy during steady-state, apneic conditions are presented. The theory shows that steady-state, top-to-bottom pleural-liquid flow creates a pressure distribution that opposes lung buoyancy. These two forces may balance, permitting dynamic lung floating, but when they do not, pleural-pleural contact is required. The animal experiments examine pleural-liquid pressure distributions in response to simulated reduced gravity, achieved by lung inflation with perfluorocarbon liquid as compared to air. The resulting decrease in lung buoyancy modifies the force balance in the pleural fluid, which is reflected in its vertical pressure gradient. The data and model show that the decrease in buoyancy with perfluorocarbon inflation causes the vertical pressure gradient to approach hydrostatic. In the microgravity analogue, the pleural pressures would be toward a more uniform distribution, consistent with ventilation studies during space flight. The pleural liquid turnover predicted by the model is computed and found to be comparable to experimental values from the literature. The model provides the flow field, which can be used to develop a full transport theory for molecular and cellular constituents that are found in pleural fluid.     

Heterologous gap junctions between oocytes and follicle cells of an insect,Dysdercus intermedius,and their potential role as ion current pathways

Summary In telotrophic insect ovaries, the oocytes develop in association with two kinds of supporting cells. Each ovary contains five to seven ovarioles. An ovariole consists of a single strand of several oocytes. At the apex of each ovariole is a syncytium of nurse cells (the tropharium), which connects by strands of cytoplasm (the trophic cords) to four or more previtellogenic oocytes. In addition, each oocyte is surrounded by an epithelium of follicle cells, with which it may form gap junctions. To study the temporal and spatial patterns of these associations, Lucifer yellow was microinjected into ovaries of the red cotton bug, Dysdercus intermedius. Freeze-fracture replicas were examined to analyze the distribution of gap junctions between the oocyte and the follicle cells. Dye-coupling between oocytes and follicle cells was detectable early in previtellogenesis and was maintained through late vitellogenesis. It was restricted to the lateral follicle cells. The anterior and posterior follicle cells were not dye-coupled. Freeze-fracture analysis showed microvilli formed by the oocyte during mid-previtellogenesis, and the gap junctions became located at the tips of these. As the microvilli continued to elongate until late vitellogenesis, gap junction particles between them and follicle cell membranes became arranged in long arrays. The morphological findings raise questions about pathways for the intrafollicular phase of the ion currents known to surround the previtellogenic and vitellogenic growth zones of the ovariole.Supported by the Deutsche Forschungsgemeinschaft (Schwerpunkt Differenzierung)     

Interaction between nitric oxide signaling and gap junctions: Effects on vascular function

Nitric oxide signaling, through eNOS (or possibly nNOS), and gap junction communication are essential for normal vascular function. While each component controls specific aspects of vascular function, there is substantial evidence for cross-talk between nitric oxide signaling and the gap junction proteins (connexins), and more recently, protein-protein association between eNOS and connexins. This review will examine the evidence for interaction between these pathways in normal and diseased arteries, highlight the questions that remain about the mechanisms of their interaction, and explore the possible interaction between nitric oxide signaling and the newly discovered pannexin channels. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Effects of 17 beta-estradiol on myometrial gap junctions and pregnancy in the rat

The effects of estradiol treatment on the development of myometrial gap junctions and premature labour were investigated using timed pregnant rats. In control animals myometrial gap junctions were infrequent between days 17 and 20 of pregnancy, but began to develop on day 21 and were at maximum frequency, size, and membrane area on day 22 during delivery. Gap junctions were completely absent from the myometrium 48 h after delivery. Animals treated with 500 micrograms 17 beta-estradiol/day starting on day 16 of pregnancy developed numerous myometrial gap junctions and delivered their pups prematurely on day 19. Similarly, treatment with 50 micrograms estradiol/day resulted in the development of myometrial gap junctions on day 20 of pregnancy and premature labour. However, treatment with various doses of estradiol up to and including 500 micrograms/day for 3 days beginning 1 day before delivery was not able to maintain the presence of myometrial gap junctions during the postpartum period. These results support the hypothesis that estradiol stimulates the development of myometrial gap junctions and that the presence of gap junctions in the myometrium is a requirement for the occurrence of term, as well as preterm labour. Furthermore, it is evident from this study that the postpartum regression of myometrial gap junctions is not dependent on the decrease in estradiol.